Project Summary/Abstract Lipid A (endotoxin) is a glucosamine-based saccharolipid that constitutes the outer monolayer of the outer membrane of Gram-negative bacteria; it is also the active component of lipopolysaccharide that causes life- threatening Gram-negative septic shock. Lipid A biosynthesis is an essential pathway conserved in virtually all Gram-negative organisms. The committed step of lipid A biosynthesis is catalyzed by UDP-3-O-(acyl)-N- acetylglucosamine deacetylase (LpxC). Because LpxC is an essential enzyme in lipid A biosynthesis and does not share sequence or structural homology with any known mammalian protein, it is an excellent target for the design of novel antibiotics. Indeed, several potent LpxC inhibitors have been discovered that display various degrees of antibiotic activity. Some of the recently discovered compounds also show time-dependent LpxC inhibition, a property that is highly desirable for an antibiotic because of the long half-life of the enzyme/inhibitor complex. A significant degree of local structural variation is likely to exist among different LpxC orthologs. Many of the potent inhibitors of Escherichia coli LpxC are relatively inactive against divergent LpxC enzymes, especially that from Pseudomonas aeruginosa, the leading cause of death in cystic fibrosis patients. CHIR-090, the most potent LpxC inhibitor discovered to date, is ineffective against multidrug-resistant Gram-negative pathogens such as Acinetobacter calcoaceticus and Burkholderia cepacia. This unusual inhibitor specificity and the lack of structural information on various LpxC/inhibitor complexes together severely hinder further optimization of existing LpxC inhibitors. The overall goal of this proposal is (1) to understand the largely unknown molecular features of LpxC underlying inhibitor specificity and time-dependent inhibition and (2) to utilize this information to improve both the potency and spectrum of inhibition for the next generation of LpxC-targeting antibiotics. This goal will be achieved by detailed structural and biochemical studies of divergent LpxC orthologs in complex with representative LpxC inhibitors, and by design, synthesis and evaluation of novel compounds based on structural insights. Project Narrative (Public Health Relevance Statement) The lack of effective treatment for multidrug-resistant Gram-negative pathogens, including strains of Pseudomonas or Acinetobacter that are resistant to all clinically available antibiotics, underscores the pressing need for antibiotics with novel mechanisms of action. Our proposed structural and biochemical studies of LpxC, an essential enzyme in lipid A biosynthesis and a novel antibiotic target of Gram-negative bacteria, will reveal the molecular basis underlying inhibitor specificity and time-dependent inhibition. Our studies have already benefited and will continue to facilitate the development of potent LpxC-targeting antibiotics against a broad spectrum of Gram-negative pathogens.